1. Field of the Invention
Embodiment 1: The concept underlying the gist of embodiment 1 of the instant invention is based on the unexpected discovery that thiophosphoryl triamide, which has been previously recognized as a urease inhibitor (U.S. SIR H25, Radel, Feb. 4, 1986; U.S. Pat. No. 4,676,822, Gautney, Jun. 30, 1987), is also capable of inhibiting nitrification of ammonium to nitrate in soil systems.
Embodiment 2: The concept underlying the gist of embodiment 2 of the instant invention is based on the unexpected discovery that this inhibitor can be used to control the concentration of ammonium in urea-containing fertilizers by effectively maintaining low levels of ammonium during early stages of plant seedling development via its urease inhibitory characteristics and by effectively maintaining high levels of ammonium nitrogen in the soil system during the later stages of plant reproductive growth.
2. Description of Prior Art
Embodiment 1: Several hundred scientific papers have been published on urease since Sumner (1926) first produced the classical octahedral crystals and showed that the enzyme was a protein. However, it was not until 1969 that Zerner's group [R. L. Blakeley, E. C. Webb, and B. Zerner, Biochemistry 8, 1984-1990 (1969)] prepared a highly purified urease with a full specific activity and in at least a 99 percent homogeneous state. They established with this preparation a reproducible molecular weight (about 590,000) and proposed that the molecule contained six subunits with asparagine as the N-terminal amino acid. Although previous work [J. F. Ambrose, G. B. Kistiakowsky, and A. G. Kridl, J. Am. Chem. Soc. 73, 1232 (1951)] had indicated that four or eight essential SH-groups were involved in the urea-hydrolysis reaction, Zerner's group could only confirm that the active site SH-groups "react slowly with N-ethylmaleimide," but they were unable to define unequivocally the number of "essential SH-groups" in the 590,000 molecular weight species. In addition, Kobashi et al. [K. Kobashi, J. Hase, and T. Komai, Biochem. Biophys. Res. Commun. 23, 34 (1966)], on the basis of inhibition by hydroxamic acids, suggested that the number of active sites in the 590,00 molecular weight species of sword bean urease was two. These results seem to be confirmed by the discovery that highly purified urease from jack bean [N. E. Dixon, C. Gazzola, R. L. Blakeley, and B. Zerner, J. Am. Chem. Soc. 97, 4131 (1975)] and from tobacco, rice, and soybean [J. C. Polacco, Plant Science Letters 10, 249-255 (1977)] contained stoichiometric amounts of nickel (two atoms per active site), demonstrating simultaneously the first biological role definitely assigned to nickel. Over the last few years, considerable effort has been made to determine the mechanism of the urease reaction. Elucidation of the mechanism of the urease reaction is complicated because the enzyme has a tendency to form polymers and isozymes changing the properties of the original monomeric enzyme and probably the mechanism of reaction [W. N. Fishbein and K. Nagarajan, Arch. Biochem. Biophys. 144, 700-714 (1971)]. In addition, the properties of soil urease differ significantly from those of ureases from other sources [J. M. Bremner and R. L. Mulvaney, Soil Enzymes, R. G. Burns, ed., Academic Press, 149-196 (1978)]; and it is much more difficult to obtain reliable kinetic data for enzymes in heterogeneous environments, such as soil, than for enzymes in homogeneous solutions. As a result most attempts to determine the mechanism of urease inhibition have been confined to non-soil systems. Attempts to demonstrate the formation of a carbamoyl-enzyme intermediate, which was postulated many years ago, have so far failed. Zerner's group [N. E. Dixon, P. W. Riddles, C. Gazzola, R. L. Blakeley, and B. Zerner, Can J. Biochem. 58, 1335-1344 (1980)] proposed a mechanism of reaction on the basis of a carbamoyl-transfer reaction and where the substrate is activated toward nucleophilic attack by O-coordination to a Ni.sup.2+ ion. Both Ni.sup.2+ ions are involved in this proposed mechanism. A second mechanism of reaction based on the determination of kinetic isotope effects [ R. Medina, T. Olleros, and H. L. Schmidt, Proc. 4th Int. Conference on Stable Isotopes, H. L. Schmidt, H. Forstel, and K. Heizinger, eds., Julich, Mar. 1981, Elsevier, Amsterdam, 77-82 (1982)] was proposed. These results indicated the existence of an enzyme-bound carbamate intermediate and demonstrated that the enzyme-Ni-substrate complex decomposes, releasing the first NH.sub.3 in a slow, rate-limiting step.
Most enzymes can be poisoned or inhibited by certain chemical reagents. There are two major types of enzyme inhibitors: irreversible and reversible. Irreversible inhibitors combine with or destroy a functional group on the enzyme molecule that is necessary for its catalytic activity. Reversible inhibitors compete with an active site or change the structure of the enzyme and generally are considered to be either competitive or noncompetitive. Competitive inhibitors compete with the substrate for binding to the active site, but once bound, cannot be transformed by the enzyme. An identifying feature of competitive inhibition is that it can be reversed by increasing the substrate concentration. Noncompetitive inhibitors do not bind at the site on the enzyme at which the substrate does; however, their binding to the enzyme alters the structure or conformation of the enzyme so that reversible inactivation of the catalytic site results.
Many urease inhibitors have been identified; however, few kinetic descriptions include the type of inhibition. The reversible and competitive inhibition of sword bean urease by a wide variety of hydroxamic acids was discovered by Kobashi et al. [K. Kobashi, J. Hase, and K. Uehara, Biochem. Biophys. Acta 65, 380-383 (1962)]. Kinetic and spectral studies performed by B. Zerner and coworkers [N. E. Dixon, J. A. Hinds, A. K. Fihelly, C. Gazzola, D. J. Winzor, R. L. Blakeley, and B. Zerner, Can. J. Biochem. 58, 1323-1334 (1980)] established that hydroxamic acids were reversibly bound to active-site nickel ions in jack bean urease. Chemical and physical studies of the enzymatically inactive phosphoramidate-urease complex provide convincing evidence that phosphoramidate binds reversibly to the active-site nickel ion [N. E. Dixon, R. L. Blakeley, and B. Zerner, Can. J. Biochem. 58, 481-488 (1980)].
The kinetics of urease inhibition by phenyl phosphorodiamidate (which demonstrates a competitive inhibition) and hydroquinone (which exemplifies a mixed inhibition mechanism) were performed by L. J. Youngdahl and E. R. Austin at the International Fertilizer Development Center (IFDC, unpublished results). A kinetic study of the soil urease inhibition by six substituted ureas, compounds which are used as herbicides, showed that all six compounds exhibited mixed inhibition characteristics (competitive and noncompetitive) [S. Cervelli, P. Nannipieri, G. Giovannini, and A. Perna, Pesticide Biochem. Physiol. 5, 221-225 (1975)].
Many other compounds have been identified as urease inhibitors. Mulvaney and Bremner [R. L. Mulvaney and J. M. Bremner, Soil Biochem. 5, 153-196 (1981)] have published an extensive review on urease inhibitors. More recently Gould and coworkers [W. D. Gould, C. Hagedorn, and R. G. L. McCready, "Urea transformations and fertilizer efficiency in soil", Advances in Agronomy 40:209-238, (1986)] described a number of urease inhibitors. These inhibitors were classified into three groups (sufhydryl reagents, hydroxamates, and structural analogs of urea and related compounds). Members of these three classes of compounds plus a fourth class (agricultural chemicals which inhibit urease) have been more extensively described by Medina and Radel (R. Medina and R. J. Radel, "Mechanisms of Urease Inhibition," submitted for publication in Ammonia Volatilization From Urea Fertilizers, B. R. Bock and D. E. Kissel, eds., to be published by the Tennessee Valley Authority).
Although a wide variety of chemicals have been shown to inhibit urease activity, none have yet been commercialized. Phenyl phosphorodiamidate (PPDA), (C.sub.6 H.sub.5 O)PO(NH.sub.2).sub.2, has probably been the most widely studied urease inhibitor in recent years and until recently was considered the best known inhibitor. Extensive work directed at commercializing PPDA has been conducted at the Tennessee Valley Authority's National Fertilizer Development Center. Most of this work has been directed at determining the compatibility of PPDA at process conditions typically encountered during urea granulation [J. Gautney, Y. K. Kim, and P. M. Gagen, I&EC Prod. R&D 23, No. 3, 483-489 (1984)], in fluid fertilizers [J. Gautney, Y. K. Kim, and A. R. Barnard, "Solubilities and Stabilities of the Nitrogen Loss Inhibitors Dicyandiamide, Thiourea, and Phenyl Phosphorodiamidate in Fluid Fertilizers," I&EC Prod. R&D 24, No. 1, 155-161 (1985)], and in solid urea during long-term bulk storage [J. Gautney, A. R. Barnard, D. B. Penney, and Y. K. Kim, "Solid-State Decomposition Kinetics of Phenyl Phosphorodiamidate," Soil Sci. Soc. Am. J. 50, 792-797 (1986)]. These studies showed that PPDA can be cogranulated with urea but decomposes relatively rapidly in solid mixtures with urea and in fluid fertilizers. As a result, commercialization of PPDA does not look promising.
Two relatively new inhibitors, thiophoshoryl triamide, (TPTA), (NH.sub.2).sub.3 PS (U.S. SIR No. H25, Radel, Feb. 4, 1986) and N-(n-butyl)-thiophosphoryl triamide (NBTPTA), [NH(CH.sub.3 CH.sub.2 CH.sub.2 CH.sub.2)]-(NH.sub.2).sub.2 PS (U.S. Pat. No. 4,530,714, Kolc et al., Jul. 23, 1985) are currently receiving considerable attention in terms of commercial development. Both of these compounds have been shown to give superiorinhibition to PPDA. The Tennessee Valley Authority has been granted a statutory invention registration on TPTA SIR No. H25, supra, and a patent on the use of TPTA in fluid fertilizers '822, supra. The inhibitor NBTPTA was patented by Allied Corporation in 1985 (U.S. Pat. No. 4,530,714, Kolc et al., Jul. 23, 1985).
Fertilizer compatibility studies with TPTA (TVA Bulletin Y-191, "New Developments in Fertilizer Technology," Oct. 1985) showed that this inhibitor can be cogranulated with urea and is relatively stable in fluid fertilizers containing urea, but like PPDA decomposes in solid mixtures with urea (R. J. Radel, J. Gautney, A. A. Randle, J. E. Cochran, R. M. Miles, H. M. Williams, B. R. Bock and N. K. Savant, "Evaluation of Thiophosphoryl Triamide as a Urease Inhibitor," paper presented at the 194th National Meeting of the American Chemical Society, Aug. 30-Sept. 4, 1987, New Orleans, La.) As a result, TPTA has considerable potential for use in urea containing fluid fertilizer but limited potential for use with solid urea. No data are currently available on the compatibility of NBTPTA with fertilizer materials. Field studies with TPTA and NBTPTA in urea containing fluid fertilizers are in progress.
Even though urease has been extensively studied for about 60 years and a large number of compounds have been identified as urease inhibitors, there are no urease inhibitors which are similarly effective as nitrification inhibitors.
The biological oxidation (nitrification) of ammonium nitrogen (NH.sub.4.sup.+) to nitrate nitrogen (NO.sub.3.sup.-) reaction (3) supra, is an energy-producing process involving a loss of eight electrons by the ammonium nitrogen and resulting in a nitrogen valence change from -3 to +5. The process is carried out in soils mainly by chemosynthetic autotrophs (Nitrobacteriaceae) in order to derive energy needed for their metabolic activities. The probable reaction sequence for the first step of nitrification (conversion of ammonium to nitrite by nitrosomonas) is: ammonia.fwdarw.hydroxylamine.fwdarw.(nitroxyl?).fwdarw.(nitrohydroxylamine )?.fwdarw.nitrite. The postulated intermediates nitroxyl (NOH) and nitrohydroxylamine (NO.sub.2 .multidot.NH.sub.2 OH) have not been positively identified, but their participation in the reaction sequence is consistent with the assumption that two electrons are involved in each intermediate oxidation step from ammonium to nitrite (R. D. Hauck, "Mode of Action of Nitrification Inhibitors", Nitrification Inhibitors-Potentials and Limitations, ASA Special Publication No. 38, 1980, American Society of Agronomy, Madison, Wis.). The second step of nitrification (conversion of nitrite to nitrate) is a single-step process which is carried out mainly by nitrobacter.
Several intensive searches have been conducted during the past 25 years to find chemicals that can inhibit the biological oxidation of ammonium to nitrate in soils. Since nitrite can be phytotoxic to plants, it is desirable that the first step of the nitrification process, the conversion of ammonium to nitrite, be preferentially inhibited because this avoids subsequent buildup of nitrite. A large number of chemicals have been found to inhibit nitrification. These chemicals include pyridines, pyrimidines, mercapto compounds, succinamides, thiazoles, triazoles, triazines, cyanamid derivatives and various thio compounds [R. D. Hauck, "Technological Approaches to Improving the Efficiency of Nitrogen Fertilizer Use by Crop Plants ", Nitrogen in Crop Production, American Society of Agronomy, Madison Wis., 52-560 (1984)]. A number of agricultural pesticides have also been shown to inhibit nitrification. The herbicides, N, N'-dimethyl-4-4-chlorophenyl urea, and ethyl and isopropyl carbamate have been shown to inhibit nitrification [M. H. Briggs and I. Segal, Life Science 2, 69-72 (1963)]. The pesticides diazion, manozeb, benzene hexachloride, pentachlorophenol, Vapam, maneb, and isodoacetic are also nitrification inhibitors [T. Nishihara, Bull. Fac. Agr. Kagoshima Univ. 12, 107-158 (1962)].
In general, most nitrification inhibitors function by either retarding the growth and/or function of nitrifiers. Inhibition can result from interference with the nitrifiers respiration and cytochrome oxidase function, by production of acid in the microenvironment, by chelation of essential metal ions, and by liberation of toxic compounds such as mercaptans, sulfoxides, and sulfones.
The biochemistry of several naturally occurring and synthetic substances which can act as nitrification inhibitors has been reviewed [J. H. Quastel, Ann. Rev. Plant Physiol. 16, 217-240 (1965)]. Methionine and some alkylmercapto amino acids delay the beginning of nitrification. This delay is believed to result from retardation of the proliferation of nitrifying organisms. Cystine and cysteine indirectly impede nitrification. Quastel [J. H. Quastel, Plant Physiol., Vol. 3 (F. C. Steward, ed.), Academic Press, New York, 671-756 (1963)], suggested that these compounds are decomposed by soil heterotrophs with the formation of sulfuric acid which decreases soil pH to a level less favorable for nitrification. Later researchers [J. M. Bremner and L. G. Bundy, Soil. Biol. Biochem. 6, 161-165 (1974)] presented evidence that cystine, cysteine, methionine, and other nonvolatile organic sulfur compounds may inhibit nitrification by decomposition to toxic carbon disulfide. Thiourea and allylthiourea are thought to inhibit nitrification by complexing with metallic cations such as Cu.sup.+2, which are needed for the process of nitrification [J. H. Quastel, Ann. Rev. Plant Physiol. 16, 217-240 (1965)]. Copper chelating agents, such as salicyladoxime and sodium diethyldithiocarbamate, also retard nitrification [H. Lees, Metabolic Inhibitors 2, (R. H. Hochester and J. H. Quastel, eds.), Academic Press, New York, 615-629 (1963)]. Addition of copper cation (Cu.sup.+2) has been shown to somewhat counteract the nitrification inhibition effect of thiourea but not that of allylthiourea [E. R. Campbell and M.I.H. Aleem, Antione van Leeuwenhoek, J. Microbiol. Serol. 31, 124-136 (1965)]. In contrast to thiourea, the inhibitors ethyl urethane [J. H. Quastel, Plant Physiol. 3 (F. C. Steward, ed.), Academic Press, New York, 671-756 (1963)], 2-chloro-6-(trichloromethly) pyridine [C.A.I. Goring, Soil Science 93, 211-218 (1962)], sodium azide, and dicyandiamide retard nitrification by acting directly on the nitrosomonas species involved in the first step of nitrification [T. Nishihara, Bull. Fac. Agr. Kagoshima Univ. 12, 107-158 (1962)]. The inhibitors, phenyl mercuric acetate, mono- and di-chlorophenyl isothiocyanates, and sodium chlorate preferentially inhibit the second step of the nitrification process, the conversion of nitrite to nitrate, [R. D. Hauck, "Synthetic Slow-Release Fertilizers and Fertilizer Amendments", Organic Chemicals in the Soil Environment 2, (C.A.I. Goring and J. W. Hamaker, eds.), Marcel Dekker, New York, 633-690 (1972)].
Although a large number of many different types of compounds have been found to inhibit nitrification (many which of are patented), only seven have been recently produced commercially for use as nitrification inhibitors. Four of these [2-amino-4-chloro-6-methyl pyridine (AM), 2-mercaptobenzothiazole (MBT), sulfathiazole (ST), and thiourea (TU)] are produced and marketed primarily in Japan.
In the United States, the compound 2-chloro-6-trichloromethyl pyridine (nitrapyrin), was commercialized as N-Serve.RTM. in 1974 and has remained on the market since that time. Another compound, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazole (ETT) was licensed in the United States as a nitrification inhibitor by Olin Corporation in 1982. Olin had previously marketed the compound as a fungicide under the trade name Terrazole. Terrazole was marketed by Olin as a nitrification inhibitor under the trade name Dwell for about one year but was withdrawn from the market when Olin's Agrichemicals Division was allegedly purchased by Uniroyal Inc., i.e., about Oct. 1 of 1983. Uniroyal apparently is not presently marketing Dwell. The compound dicyandiamide (Dd, DCD, Dicyan) has received much attention in the United States in recent years because it appears to offer some advantages over nitrapyrin and ETT. Both nitrapyrin and ETT have relatively high vapor pressures. These vapor pressures prevent cogranulation of nitrapyrin and ETT with solid fertilizers such as urea. Their use with nitrogen solutions is also limited because of volatilization losses during fertilizer processing and application. As a result, nitrapyrin and ETT are used primarily with anhydrous ammonia; however, even then special precautions must be taken. Nitrapyrin can be tank-mixed with anhydrous ammonia but has a maximum recommended shelf life of three weeks (Dow Chemical Company USA. "N-Serve Nitrogen Stabilizer," Technical Information Bulletin, Ag-Organics Department, Midland, Mich.). ETT cannot be tank-mixed with anhydrous ammonia and therefore must be applied simultaneously from a separate tank. DCD, on the other hand, has very little vapor pressure and as a result can be cogranulated with solid fertilizers, such as urea, without significant losses of inhibitor [J. Gautney, Y. K. Kim, and P. M. Gagen, Ind. Eng. Chem. Prod. Res. Dev. 23, No. 3, 483-489 (1984)]. Dicyandiamide is also stable in anhydrous and aqueous ammonia solutions [J. Gautney, Y. K. Kim, and P. M. Gagen, Ind. Eng. Chem. Prod. Res. Dev. 24, No. 4, 645-650 (1985)].
Dicyandiamide is produced and marketed in Japan and more recently is being produced in West Germany and test-marketed in the United States as a slow-release nitrogen fertilizer. It appears that dicyandiamide is currently not registered as a nitrification inhibitor in the United States. Although DCD offers a number of advantages over nitrapyrin and ETT in terms of fertilizer compatibility, like nitrapyrin and ETT, it is relatively expensive to use, especially for use with fertilizer materials.
An intensive search of the literature has revealed only two instances in which phosphorus-containing compounds have shown nitrification inhibition properties. U.S. Pat. No. 4,315,762, Evrard, Feb. 16, 1982, describes the use of aluminum Tris(O-ethyl phosphonate) I. These materials which have a covalent aluminum-phosphorus bond are of the formula ##STR3##
The second instance describes the results of a series of tests on nine phosphoroamides which were found effective as urease inhibitors [J. M. Bremner, G. W. McCarty, J. C. Yeomans, and H. S. Chai, Commun. Soil Sci. Plant Anal. 17, 369-84]. Of the nine phosphoroamides tested, only two exhibited minimal inhibitory activity, the trichloroethyl phosphorodiamidate (II) and n-butyl-phosphorothioic triamide III). These phosphoroamides have the structures shown infra. This later compound appeared to inhibit nitrification only at very high rates of application (10 to 20 times), and to a much lower extent (25 to 33 percent) when compared to nitrapyrin or etridiazole (both commercial inhibitors) in terms of inhibitor to soil ratios and incubated at 20.degree. C. When the incubation temperature is raised to 30.degree. C., the inhibition is even lower (4-12 percent). ##STR4##
Embodiment No. 2: There is little consensus concerning optimum ammonium/nitrate ratios for crop growth. This is not suprising since the ammonium/nitrate ratio available to roots affects physiological processes by a number of mechanisms. This area of concern was recently reviewed by B. R. Bock (1986). Increasing cereal yields with higher ammonium/nitrate ratios:review of potentials and limitations [J. Environ. Sci. Health, A21:723-758]. A summary of this review is given below.
Hageman [R. H. Hageman, Ammonium versus nitrate nutrition of higher plants, 67-85 (1984)] and Hauck [R. D. Hauck (ed.), Nitrogen in crop production, American Society of Agronomy, Madison, Wis.] reviewed several of these mechanisms and their physiology.
First, the ammonium/nitrate ratio in the root medium affects nitrogen fertilizer adsorption rates by roots. Relative rates of ammonium and nitrate absorption are affected by temperature, pH, ionic composition of the soil solution, and stage of plant growth, making generalizations about ammonium/nitrate ratio effects on nitrogen fertilizer absorption rates difficult. However, there appears to be some potential for increasing cereal yields by manipulating ammonium/nitrate ratios to enhance nitrogen fertilizer uptake rates, particularly during reproductive growth stages.
Second, since nitrogen fertilizer is absorbed in relatively large quantities compared with other inorganic plant nutrients, the ionic form of nitrogen fertilizer absorbed affects uptake of other cations and anions. This affects the level of inorganic nutrients absorbed by plant roots and also the metabolic requirements for regulating ionic balance and pH within the plant.
Third, the rhizosphere tends to become acidic when ammonium is the dominant form absorbed by roots and tends to become alkaline when nitrate is the dominant nitrogen fertilizer form absorbed. These pH changes can affect plant growth either directly via H.sup.+ ions or by affecting the availability of plant nutrients or toxic elements to plant roots.
A fourth mechanism relates to the potential for toxic levels of unassimilated ammoniacal nitrogen fertilizer within plants. Unassimilated ammoniacal nitrogen fertilizer is present in plants as both ammonium and NH.sub.3 (aq) with relative levels determined by the following equilibrium: EQU NH.sub.3 +H.sub.2 O.revreaction.NH.sub.4.sup.+ +OH.sup.- ( 4)
Both species can be toxic to plants [H. M. Reisenhauer, "Absorption and Utilization of Ammonium Nitrogen by Plants", 157-170 (1978) and, D. R. Nielsen and J. G. MacDonald (eds.), "Nitrogen in the Environment", Vol. 2. Soil-plant nitrogen relationships, Academic Press, New York ]. The term ammonium toxicity will be used hereafter to designate direct adverse effects of unassimilated ammonium and/or aqueous NH.sub.3 on plant growth.
When external ammonium is readily available, ammonium absorption by roots can be more rapid than ammonium assimilation processes which detoxify the absorbed ammonium. This can result in ammonium toxicity because plants tolerate only relatively low levels of unassimilated ammonium. More rapid uptake than assimilation of ammonium is a problem, particularly when low light intensities or other environmental factors limit production of carbon skeletons required for assimilation. In contrast, nitrate can assimilate in plants to relatively high levels without adverse effects on growth. Physiologically regulated mechanisms ensure that toxic ammonium levels do not develop from reduction of nitrate.
A fifth mechanism relates to energy requirements for nitrogen fertilizer assimilation. Ammonium assimilation requires less energy than nitrate assimilation in nonchlorophyllous tissue such as roots or in shaded chlorophyllous tissue of organs. However, in chlorophyllous organs (primarily leaves), nitrate assimilation may require no more energy than ammonium assimilation in terms of carbohydrate reserves.
Raven and Smith [J. A. Raven and F. A. Smith, "Nitrogen Assimilation and Transport in Vascular Land Plants in Relation to Intracellular pH Regulation," New Phytol. 76:415-431 (1976)] reviewed several aspects of a sixth mechanism, pH regulation, within the plant. The form of nitrogen fertilizer affects both internal pH and metabolic requirements for regulating pH. Nitrate assimilation generates OH.sup.- ions, mainly in shoots where pH is regulated by a biological pH-stat that produces organic acids from neutral precursors. Hydrogen ions from these acids neutralize alkalinity from nitrate assimilation. The resulting organic acid anions are either stored in shoots to maintain ionic balance or transported to roots with subsequent release of OH.sup.- ions into the soil solution. Up to 10 percent of the organic carbon in the plant can be involved in organic acid anion storage resulting from operation of the pH-stat. Operation of the pH-stat places significant demands on carbon and energy economies of the plant and can cause problems in regulating cell turgor and volume. Alternatively, ammonium assimilation generates H.sup.+ ions, mainly in roots where pH is regulated by excreting H.sup.+ ions into the soil solution. The latter type of internal pH regulation places minimal demands on the plant.
Tsai, et al. [C. Y. Tsai, H. L. Warren, and D. M. Huber, "The Kernel Nitrogen Fertilizer Sink as a Biological Yield Component in Maise," Proceedings of 37th Annual Corn and Sorghum Research Conference, 52-66 (1982)] suggest a seventh mechanism arising from enhanced sucrose movement from leaves to roots with ammonium assimilation compared with that for nitrate assimilation. Ammonium assimilation typically occurs immediately after absorption by roots as a means of detoxifying ammonium and requires a readily available supply of organic acids which are derived from sucrose. This demand is thought to enhance sucrose movement from leaves to roots and thereby reduce "feedback" inhibition of photosynthesis. However, excess ammonium can reduce plant growth by depleting carbon required for growth, particularly during the seedling stage when photosynthetic capacity is low or when environmental conditions severely limit photosynthesis. In contrast, nitrate assimilation results in less carbon translocation from leaves to roots, providing less potential for either reducing "feedback" inhibition of photosynthesis or depleting carbon reserves required for growth. This is because nitrate assimilation occurs to a large extent in shoots, and significant quantities of nitrate are stored as nitrogen fertilizer reserves before being assimilated.
An eighth possible mechanism relates to nitrate reductase activity as a growth limiting factor in some situations. Olsen [S. R. Olsen, "The Role of Organic Matter and Ammonium in Producing High Corn Yields," Proceedings of International Symposium on Peat in Agriculture and Horticulture, Bet Dagan, Israel (1983)] concluded that when plants rely mainly on nitrate as a nitrogen fertilizer source, nitrate reduction can be the rate-limiting step in the supply of reduced nitrogen fertilizer to plants and, in turn, a growth limiting factor. Shaded leaves and high-yield agriculture were cited as situations where nitrate reductase activity likely limits yields. In these situations, substituting some ammonium for nitrate in the root medium may remove nitrate reduction as a growth-limiting step in the supply of reduced nitrogen fertilizer for the plant. Shaner and Boyer [D. L. Shaner and J. S. Boyer, "Nitrate Reductase Activity in Maize (Zea mays L.) Leaves," I. "Regulation of Nitrate Flux," Plant Physiol. 58:499-5041976 (1976)] found that the flux of nitrate to leaves rather than the nitrate reductase enzyme is more often the limiting factor in nitrate assimilation, in which case ammonium could serve as a supplemental source of reduced nitrogen fertilizer to the leaves.
Finally, ammonium/nitrate ratios absorbed by plant roots affect the biochemistry of plants in several respects not mentioned above, including the activity of several enzymes [Hageman, ibid., S. S. Sham, and R. C. Huffaker, "Nitrogen Toxicity in Plants," 97-118 (1984)] and [R. D. Hauck (ed.). "Nitrogen in Crop Production," American Society of Agronomy, Madison, Wis.].
Relatively little is known about the optimum ratios necessary for maximum crop yields. Recent studies have shown, however, that high ammonium/nitrate ratios during early plant growth can result in decreased yields [B. R. Bock, "Agronomic Difference between Nitrate and Ammoniacal Nitrogen," Proceedings of the 37th Annual Meeting Fertilizer Industry Round Table, 105-106 (1987)]. In a field study using urea and DCD, ratios as high as 31:1, within 11 days of planting, i.e., during at least a portion of the germination stage, significantly reduced yields at harvest (by about 10 percent), even though ratios remained high during the remaining portion of the growing season.
Although no studies have documented the exact ratios needed for optimum plant growth and yield, it is postulated that high ammonium/nitrate ratios during the reproduction, or in the case of grain crops, during the grain-filling stage are necessary for maximum yields (Bock, ibid, p.11 1986).
From the above information, it is clear that although a large number of compounds have been identified and are patented separately as urease inhibitors or as nitrification inhibitors, there is no single inhibitor which meets all the needs of American agriculture. The present invention relates to the discovery that there are specific compounds which meet such multiple needs, as such dual-purpose nitrification and urease inhibition.